Summary The overall goal of this project is to develop, validate, and commercialize the first-ever hemoglobinopathies panel kit on Oxford Nanopore Technologies? NP system, covering deletions, duplications and mutations in the alpha and beta globin genes associated with thalassemias. Our panel will detect phasing of silent carriers in alpha-thalassemia and assess the risk for both alpha- and beta-thalassemia, despite a negative result from conventional methods of testing. No commercially-available kit offers this range of capabilities. These tasks will accomplished by leveraging three enabling technologies: (i) Asuragen?s proprietary AmplideX PCR chemistries for the amplification of complex DNA and DNA barcode implementation; (ii) Oxford Nanopore Technologies? MinION system, the first available NP platform for direct long-read sequencing for the analysis of long and challenging stretches of DNA; and (iii) Asuragen?s proprietary haplotype informatics phasing algorithms. Standard approaches for screening require a variety of cumbersome sequential tests that do not identify all carriers. In contrast, our comprehensive test will assess carrier risk for alpha- and beta-thalassemias, ascertain carrier status for alpha-thalassemia with relevant in-phase mutations, identify the large disease-causing deletions found in alpha and beta globin genes, and provide clinically-relevant copy number detection and phasing of the four alpha hemoglobin genes. The proposed test will further exploit this phasing approach to resolve sequence redundancy between the two genes that encode alpha hemoglobin (HBA1 and HBA2) and accurately call >200 mutations within the HBB gene that causes beta-thalassemia. Complementary wetware and software advancements will be integrated to overcome workflow, platform, and assay-related challenges and establish feasibility for the PCR/NP phased-sequencing approach for hemoglobinopathies. Asuragen?s success in building end-to-end solutions for complex PCR and NGS assays by combining breakthrough chemistries, simple workflows, and sophisticated software suites, set a solid foundation for this project to develop a modular 3rd gen NGS solution for carrier screening. The specific aims are: Aim 1: Establish a scalable, and robust PCR-based enrichment method for generating multiplexed and barcoded HBA1, HBA2 and HBB gene cluster libraries, that is compatible with high-throughput and high- resolution phased-sequencing on the ONT platform. Aim 2: Develop a novel NP sequencing analysis pipeline that enables comprehensive and automated phasing of HBA1 and HBA2 and accurate detection of deletions and mutations of HBA1, HBA2 and HBB gene clusters known to cause hemoglobinopathies. Aim 3: Demonstrate feasibility for a unified single-day workflow and analysis pipeline at a 96-plex scale for the MinION system and/or 10-plex for the Flongle system, using gDNA from a clinically relevant set of residual blood specimens that span the range of hemoglobinopathy genotypes.